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Evaluation of candidate reference genes for quantitative expression studies in Asian seabass (Lates calcarifer) during ontogenesis and in tissues of healthy and infected fishes.

Abstract Quantitative real-time PCR (qRT-PCR), used to determine the gene expression profile, is an important tool in functional genomic research, including fishes. To obtain more robust and meaningful result, the best possible normalization of the data is of utmost significance. In the present study, we have evaluated the potential of five commonly used housekeeping genes i.e., elongation factor 1-α (EF1A), β-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-2-Microglobulin (B2M) in normal physiological conditions, developmental stages and in response to bacterial infection in Asian seabass, Lates calcarifer (Bloch), an important food fish cultured in the Asia-Pacific region. The expression levels of these five genes were estimated in 11 tissues of normal seabass juveniles, 14 embryonic and larval developmental stages and six tissues of Vibrio alginolyticus-challenged animals. Further, the expression stability of these genes was calculated based on three algorithms i.e. geNorm, NormFinder and BestKeeper. The results showed that although there are tissue-specific variations for each gene, ACTB and EF1A are the most stable genes across the tissues of normal animals. However, in bacteria-challenged animals, EF1A and 18S were found to be the best reference genes for data normalization. The expression of all the genes tested showed an increasing trend in developmental stages and the increase was significant at blastula stage. Among the five genes tested, EF1A and ACTB were found to be the genes with least variation and highest stability across the developmental stages. This forms the first report on validation of housekeeping genes in L. calcarifer, in the context of ontogenic development and in response to infection.
PMID
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Authors

Mayor MeshTerms
Keywords
Journal Title indian journal of experimental biology
Publication Year Start




PMID- 28699725
OWN - NLM
STAT- MEDLINE
DA  - 20170712
DCOM- 20170801
LR  - 20170801
IS  - 0019-5189 (Print)
IS  - 0019-5189 (Linking)
VI  - 54
IP  - 9
DP  - 2016 Sep
TI  - Evaluation of candidate reference genes for quantitative expression studies in
      Asian seabass (Lates calcarifer) during ontogenesis and in tissues of healthy and
      infected fishes.
PG  - 597-605
AB  - Quantitative real-time PCR (qRT-PCR), used to determine the gene expression
      profile, is an important tool in functional genomic research, including fishes.
      To obtain more robust and meaningful result, the best possible normalization of
      the data is of utmost significance. In the present study, we have evaluated the
      potential of five commonly used housekeeping genes i.e., elongation factor
      1-alpha (EF1A), beta-Actin (ACTB), 18S ribosomal RNA (18S),
      glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-2-Microglobulin (B2M)
      in normal physiological conditions, developmental stages and in response to
      bacterial infection in Asian seabass, Lates calcarifer (Bloch), an important food
      fish cultured in the Asia-Pacific region. The expression levels of these five
      genes were estimated in 11 tissues of normal seabass juveniles, 14 embryonic and 
      larval developmental stages and six tissues of Vibrio alginolyticus-challenged
      animals. Further, the expression stability of these genes was calculated based on
      three algorithms i.e. geNorm, NormFinder and BestKeeper. The results showed that 
      although there are tissue-specific variations for each gene, ACTB and EF1A are
      the most stable genes across the tissues of normal animals. However, in
      bacteria-challenged animals, EF1A and 18S were found to be the best reference
      genes for data normalization. The expression of all the genes tested showed an
      increasing trend in developmental stages and the increase was significant at
      blastula stage. Among the five genes tested, EF1A and ACTB were found to be the
      genes with least variation and highest stability across the developmental stages.
      This forms the first report on validation of housekeeping genes in L. calcarifer,
      in the context of ontogenic development and in response to infection.
FAU - Paria, Anutosh
AU  - Paria A
FAU - Dong, Jie
AU  - Dong J
FAU - Babu, P P Suresh
AU  - Babu PPS
FAU - Makesh, M
AU  - Makesh M
FAU - Chaudhari, Aparna
AU  - Chaudhari A
FAU - Thirunavukkarasu, A R
AU  - Thirunavukkarasu AR
FAU - Purushothaman, C S
AU  - Purushothaman CS
FAU - Rajendran, K V
AU  - Rajendran KV
LA  - eng
PT  - Journal Article
PL  - India
TA  - Indian J Exp Biol
JT  - Indian journal of experimental biology
JID - 0233411
RN  - 0 (Actins)
RN  - 0 (Peptide Elongation Factor 1)
RN  - 0 (RNA, Ribosomal, 18S)
RN  - EC 1.2.1.- (Glyceraldehyde-3-Phosphate Dehydrogenases)
SB  - IM
MH  - Actins/genetics
MH  - Animals
MH  - Bacterial Infections/genetics/*veterinary
MH  - Fish Diseases/*genetics
MH  - Glyceraldehyde-3-Phosphate Dehydrogenases/genetics
MH  - Peptide Elongation Factor 1/genetics
MH  - Perciformes/*genetics/microbiology
MH  - RNA, Ribosomal, 18S/genetics
MH  - Real-Time Polymerase Chain Reaction
EDAT- 2017/07/13 06:00
MHDA- 2017/08/02 06:00
CRDT- 2017/07/13 06:00
PST - ppublish
SO  - Indian J Exp Biol. 2016 Sep;54(9):597-605.